Process for the biological production of alpha,omega-alkanedioic acid

ABSTRACT

The improvement in a process for the production of alpha,omegaalkanedioic acid of 10 to 14 carbon atoms in a mixture comprising n-alkane of 10 to 14 carbon atoms, culture medium and a mutant of Corynebacterium capable of producing at high yield and conversion alpha,omega-alkanedioic acid which comprises operating the process in the presence of an inducer, i.e., a blend of substantially branched saturated aliphatic hydrocarbons of 12 to 22 carbon atoms, the blend having a boiling point of from 150* to 255*C. and/or a blend of n-alkanes having from 15 to 24 carbon atoms with the provisoes that initially the ratio of volume of the inducer to the n-alkane of 10 to 14 carbon atoms is from 3:1 to 1:3 and the volume of the n-alkane of 10 to 14 carbon atoms is from 1 to 50 percent of the total volume of the culture medium, and n-alkane of 10 to 14 carbon atoms.

United States Patent [1 1 Dahlstrom et al.

[ PROCESS FOR THE BIOLOGICAL PRODUCTION OF ALPHA, OMEGA-ALKANEDIOIC ACID [75] Inventors: Robert Victor Dahlstrom; James Herbert Jaehnig, both of Manitowoc, Wis.

[73] Assignee: E. I. du Pont de Nemours and Company, Wilmington, Del.

[22] Filed: Dec. 30, 1971 [21] Appl. No.: 214,380

[52] U.S. Cl 195/28 R [51] Int. Cl C12d 1/02 [58] Field of Search 195/28 R, 34, 30, 195/1 14 [56] References Cited UNITED STATES PATENTS 3,483,083 12/1969 Elson et al. 195/30 FOREIGN PATENTS OR APPLICATIONS Nov. 20, 1973 Primary ExaminerLionel M. Shapiro Assistant Examiner-R. B. Penland Attorney-William A. Hoffman [5 7] ABSTRACT The improvement in a process for the production of alpha,omega-alkanedioic acid of 10 to 14 carbon atoms in a mixture comprising n-alkane of 10 to 14 carbon atoms, culture medium and a mutant of Corynebacterium capable of producing at high yield and conversion alpha,omega-alkanedioic acid which comprises operating the process in the presence of an inducer, i.e., a blend of substantially branched saturated aliphatic hydrocarbons of 12 to 22 carbon atoms, the blend having a boiling point of from 150 to 255C. and/or a blend of n-alkanes having from 15 to 24 carbon atoms with the provisoes that initially the ratio of volume of the inducer to the n-alkane of 10 to 14 carbon atoms is from 3:1 to 1:3 and the volume of the nalkane of 10 to 14 carbon atoms is from 1 to 50 per cent of the total volume of the culture medium, and n-alkane of 10 to 14 carbon atoms.

7 Claims, No Drawings PROCESS FOR THE BIOLOGICAL PRODUCTION OF ALPHA, OMEGA-ALKANEDIOIC ACID This invention relates to an improved process for the production of alpha, omega-alkanedioic acid from nalkanes through the use of mutants of Corynebacterium. Particularly, this invention relates to utilizing an inducer in the process during the fermentation, the inducer comprising substantially branched saturated aliphatic hydrocarbons of 12 to 22 carbon atoms and- /or a blend of n-alkanes of 15 to 24 carbon atoms.

1t is known that alpha,omega-alkanedioic acid can be prepared by allowing mutants of Corynebacterium 7E1C( ATCC-l9067) to ferment with n-alkanes. Processes which have been utilized for such fermentation have not achieved commercially attractive yields andconversions of acid. Therefore, improvement in the processes was required to improve such yields and conversions.

Such an improvement has been found; The improvement comprises operating the process for the production of alpha,omega-alkanedioic acid of 10 to 14 carbon atoms in a mixture comprising n-alkane of 10 to 1 -4 carbon atoms, culture medium and a mutant of Corynebacterium capable of producing alphapmegaalkanedioic acid at high yields and conversions in the presence of an inducer selected from the class consisting of a blend comprising substantially branched saturated aliphatic hydrocarbons of 12- to 22 carbon-atoms, the blend having a. boiling point of from l 5O -255 C., a blend comprising n-alkanes havingfrom 15 to 24icarbon atoms and mixtures thereof with the-provisoes that initially the ratio of the volume of the inducer tothenalkane of 10 to 14 carbon atoms'isfrom 3:1 to'l :3'and* the volume of the n-alkane of l=to l4carboncat'oms is from 1 to 50 percent of the totalzvolume of the culture medium and n-alkane of 10 to 1'4 carbon-atoms.

The preferred amount of-n-alkane of tol4carbon atoms present relative to the totalvolume oftheculture medium and n-alkane of- 10 to 14 carbon atomsis-from carbon atoms, in particular; a blend comprised mainly branched saturated aliphatic hydrocarbonsof' l-2to 22 v of hydrocarbons of at least l4-carbonatomsz The preferred blend boils at from 196 to 250C. A preferred' blend of n-alkanes is a blend of n-alkanes of at'leastl' carbon atoms.

A culture medium which is useful in thepresentinvention is aqueous and comprises 1) 3 to 35 grams per liter of a member selected from the class consisting of:

alkali metal nitrate, calciumnitrate, magnesium niacetate, calcium acetate, magnesium acetate, stron-' tium acetate, beryllium acetate and mixtures thereof;-

4) a nutrient source.

The amount of metal nitrate utilized in the culture medium depends on the mutant of Corynebacteriumr which is used. A relatively large amount'of nitrate is required for the bacteria-utilizes it as the sole source of nitrogen. Preferably 6 to 30 grams per liter of the metal nitrate is present in the culture medium. The preferred:

ntirates are the alkalimetal nitrates, in particular-so dium or potassium. The most preferred concentration of the metal nitrate in the culture medium is 12 to 25 grams per liter.

The alkali metal phosphate is used as a buffer component in the culture medium. A sufficient amount of the phosphate is present in the culture medium to cause it to have a pH of from 6 to 9. Therefore, the amount utilized will vary according to the other components in the culture medium. The preferred amount of alkali metal phosphate in the culture medium is l to 6 grams per liter. Preferred phosphates are K l'lPO and NaI-LPO," H O.

The metal acetate is critical as a component in the culture medium in that it significantly increases the yield of the alpha,omega-alkan'edioic' acid when the culture medium is used in the process of this invention. The" preferred concentration for the metal acetate is from 2.5 to about 10 grams per liter. Preferred acetates are the alkali metal acetates, in particular, sodium and potassium.

The type of nutrient source in the culture medium is not critical. Examples of useful nutrient sources are tom-atojuice' brothsolids, molasses plus yeast extract or cottonseed meal. The first two types of nutrient source are preferred. The amount of nutrient source utilized is normally from 1' to 5 grams per liter, preferably around about 2 grams per liter of the culture medium.

The balance of the'culture medium is made up of water. Thiscan be tap water or distilled water. Other com ponents-can 'be' present in'the culture medium if they 'present for they erode the ability of the medium to" causethe'bacte'rium to accumulate acid. Copper compounds-can be included in the medium. However, they tend to give no advantage but instead result in reducedacid' production;

The culture medium can contain emulsifier. This is" not'requiredbutdoes normally render the culture me'' dium'moreconducive to manipulation. Useful emulsifiers are substances such as Ethofats (Armour and Company, (3hicago, Illinois) which is monofatty or rosin'acidesters' of polyo'x'yethylene glycols having the general formula 0 also cmcmmm Examples of other nutrient'type additives which can be included 'in' the culture medium are amino acids, vi-' tamins, glucose, sugar, adipic acid, butyric acidand glutaric 'acidi The culture medium which is'discussed above is the medium which is usedin theprocessfor preparing the alpha,omega-'alkanedioic acid in the full scale fermentation but is normally not used in the pregrowth of the bacteria. Pregrowthofthe bacteria can be accomplished by placing a lyophile culture of freeze-dried bacteria =on-a tomato juice agar slant which contains proteins and carbohydrates, i.e., tomato juice extract and glucose. The bacteria grows for from 24 to 48' hours at'3 7C. on theslant.'A*loopful of the bacteria is then transferred to a large flask such as a 125 ml. Erlenmyer for pregrowth. In the pregrowth stage the bacteria normally grows on a solution comprising weight- /volume percent molasses, preferably cane, 0.5 weight- /volume percent yeast extract, 0.15 weight/volume percent K HPO 0.06 weight/volume percent Nal-l PO H O and water. Weight/volume percent means grams/- milliliters in percent terms, i.e., 5 grams in 100 mls. is 5 weight/volume percent.

The pregrowth flask is maintained at about 37C. and vibrated at 220 rpms. on a shaker with a 1 inch throw for 24 hours. The volume of the material in the initial pregrowth flask is normally 25 ml. although this is not critical. The pregrowth medium plus bacteria is built up by successive spiking of larger and larger volumes. When a pregrowth volume is obtained which is about percent of the total volume of materials to be used to make the alpha,omega-alkanedioic acid, the pregrowth stage is completed. At this point, the pregrowth medium should contain at least l X 10 organisms per ml. Normally, all the pregrowth medium is used to inoculate the culture medium and n-alkane.

The invention involves an improvement of the normal process for the production of alpha,omegaalkanedioic acid. The normal process for the production of alpha,omega-alkanedioic acid of 10 to 14 car bon atoms comprises (1) introducing a mutated strain of Corynebacterium capable of producing at high yield and conversion alpha,-omega-alkanedioic acid of 10 to,

14 carbon atoms into a composition which has a'pl-l of from 6 to 9 and which comprises 1 to 50 percent by volume of n-alkane of 10 to 14 carbon atoms and a complemental amount of the culture medium above; (2) maintaining the composition at a temperature of between about to 45C. for 48 to 96 hours and (3) separating the alpha,omega-alkanedioic acid from the resulting mixture.

In the recitation of the process above it is stated that the Corynebacterium mutant is capable of producing at high yields and conversions the alpha,omegaalkanedioic acid. When the process of this invention is utilized, this statement means to produce alpha,omegaalkanedioic acid at yields and conversions such that the percentage of the n-alkane converted to the alpha,omega-alkanedioic acid is at least 12.0 mole percent; preferably at least 19 mole percent. The amount of n-alkane converted to alpha,omega-alkanedioic acid (on a mole percent basis) following the process of the present invention ranged from to percent with some bacterium. This is calculated by multiplying the yield times the conversion. Conversion is defined as the percentage amount in moles of the n-alkane converted to other than the n-alkane without consideration as to what it is converted to while yield means the percentage amount in moles of the n-alkane converted which is in the form of the alpha,omega-alkanedioic acid. Normally when conversion is high, yield is lower and vice versa. Therefore, these can vary up and down but the product of the two is always greater than the aboverecited percentages.

Some preferred mutated strains of the Corynebacterium are on file with the American TypeACuIture Collection (ATCC), 12301 Parklawn Drive, Rockville, Md. 20852. These strains are referred to throughout by their ATCC number. N

The organism tentatively assignedto the genus Corynebacterium was first isolated by J. W. Foster (.1. Bacteriology 85, 859 (1963). The original culturewas pink but after a series of mutations with chemical and physical mutagens the culture lost the capability of producing a pigment. Theorganism is a nonmotile, gram rod with metachromaticgranules. Snapping division produces angular and palisade arrangement of cells. Older cells often lose gram characteristic with granules still evident. The bacterium is in obligate aerobe and utilizes nitrates as sole sourceIof nitrogen. The bacterium rods are 0.3 to 0.4 by 0.7 to 0.8 micron. They grow well on most common laboratory media, but grow better on the culture medium recited above. Colonies of bacteria on tryptone glucose agar are dry, granular and small. Optimum temperature of growth is 37C.

The mixture inoculated by the Corynebacterium in the normal process comprises the composition, i.e., the n-alkane of 10 to 14 carbon atoms and the culture medium as discussed above and the inducer, i.e., the blend of substantially branched saturated aliphatic hydrocarbons of 12 to 22 carbon atoms and/or a blend of nalkanes of 15 to 24 carbon atoms. Normally, the Corynebacterium mutant is added as part of an inoculum as is described above. This inoculum is usually initially present in the reactor vessel at about 5 to 20 percent by volume of the composition, inoculum and inducer.

The pH of the mixture of the n-alkane of 10 to 14 carbon atoms, culture medium and inducer should be from 6 to 9 initially with the preferred range being from 7 to 8. The most preferred range is from 7 to 7.5. The temperature of the contents of the reactor vessel should be from 20 to 45C. both at the time the bacterium is added and during fermentation. The preferred range is 25 to 35C. The fermentation is normally continued for up to 96 hours. High yields and conversions are not obtained until about 48 hours and after 72 hours diminishing returns set in.

One method of separating the alpha,omega-alkanedioic acid from the resulting mixture is to (1) remove the cells and excess hydrocarbon from the phase by phase separation, (2) adjust the pH of the aqueous layer to 2.0 with concentrated sulfuric acid, (3) filter out the precipitate, (alpha,omega-alkanedioic acid) and (4) wash precipitate with water. The purity of the acid is normally 50 to 65 percent by weight when this procedure is followed.

Mutants of the Corynebacterium which are useful in the process can be prepared by the standard techniques of (1) high energy radiation; (2) chemical mutation; (3) lethal synthesis and combinations thereof. The two former techniques are preferred. The mutants referred to herein were prepared by one or a combination of these three methods.

The process is useful in preparing the alpha,-omegaalkanedioic acid. The acids are useful as intermediates in the preparation of polyamides which have low water absorption and good stiffness and which can be formed into fibers.

The following Examples are meant to illustrate but not to limit the invention. Parts and percentages are by weight unless otherwise specified.

EXAMPLE I Molasses yeast broth having the following composition was prepared:

0.15 weight/volume percent of K,HPO

0.06 weight/volume percent of NaH,PO -H,O,

5 weight/volume percent of cane molasses,

0.5 weight/volume percent yeast extract, and

remainder water.

The molasses yeast broth was adjusted to a pH of 7.0 to 7.1 with NaOH, 60 ml. was transferred to a 300 ml. shaker flask and autoclaved for minutes at 121C. A loopful of cells of a mutant of Corynebacterium sp. 01785 (ATCC 21747) from a fresh growth on a tomato juice agar slant was transferred to the sterile molasses yeast broth. The broth was incubated at 37C. for 24 hours in a 1 inch throw rotary shaker at 220 rpms. This broth was used as an inoculum for 500 mls. of molasses yeast broth in a 2-liter shaker flask. The 2-liter shaker flask was incubated for 24 hours at 37C. in a rotary shaker at 240 rpms. Contents of two of the 2- liter shaker flasks were utilized as the inoculum for a fermenter jar. The cell count in the inoculum was 3 X 10 cells per ml. while the pH of the inoculum was 7.2.

The fermenter jars were prepared as follows:

To a 14-liter jar was added 2100 mls. oftap water; 40 grams sodium acetate; grams tomato juice broth solids; 40 mls. of Ethofats, mono-fatty or rosin acid esters of polyoxyethylene glycols; 1,000 mls. dodecane; 1,000 mls. ofS0ltrol 170 (Phillips Petroleum Co.), a blend composed of substantially branched saturated aliphatic hydrocarbons of 14 to 22 carbon atoms boiling in the range of 196 to 250C.; and 5,000 mls. of a doubly concentrated salts and buffer medium prepared as fol+ lows:

The following ingredients were added to the distilled water in their respective concentrations:

0.04 weight/volume percent MgSO '7H O 0.3 weight/volume percent K HPO 0.12 weight/volume percent in an NaH PO 'I-I O 5 weight/volume percent NaNO 0.003 weight/volume percent CaCl Stock Solution Concentration 0.2 volume percent of stock solution FeSO containing ethylenediamine tetraacetic acid chelating agent 0.2 volume percent of stock solution MnSO 'H,O 0.2 volume percent of stock solution H 150, 0.2 volume percent of stock solution Na,MoO -2H,O

0.125 g/100 mls.

80 rngs/lOO mls.

5 mgs/100 mls.

43 mgs/lOO mls. 0.2

volume percent of stock A solution K1 20 mgs/lOO mls.

0.2 volume percent of stock solution ZnSO.'7H,O

into. the fermenter. The fermenter was operated under these conditions for 91 hours. The millimoles of dodecanedioic acid produced per liter of reactants in the fermenter was 97.8 (gas chromatography). The

percent (mole) conversion was 77 and the percent (mole) yield was 33.

EXAMPLE 11 A loopful of Corynebacterium sp. 234-30 (ATCC 21745) cells was transferred from a two-day agar slant growth on tomato juice agar to molasses yeast broth (mls. in a 300 ml. shaker flask) prepared as in Example l. The broths were incubated for 24 hours at 37C. in a 1 inch throwrotary shaker at 220 rpms and used as an inoculum to second stage broth (10 volume percent inoculu'm equals 25 mls. to 250 mls. fresh molasses yeast broth in one liter shaker flask). Second stage broth was incubated for 27 hours at 37C. in a liter shaker flask at 240 rpms. This then was used as the inoculum to the main fermentation media test flask.

The main test flasks were prepared as follows: doubly concentrated salts and buffer medium prepared as in Example 1, was diluted to 65 percent of its original concentration with distilled water. To this medium was added 0.3 weight/volume percent tomato juice broth solids and 0.5 weight/volume percent sodium acetate. 20 Mls. of this solution was measured into 300 ml. shaker flask with 0.1 ml. Ethofats emulsifier, 2.5 mls. n-dodecane and 2.5 mls. Soltrol 170 (a blend of substantially branched saturated aliphatic hydrocarbons of 14 to 22 carbon atoms, boiling point 196 to 250C.). The flasks were autoclaved after being gauze covered.

5 M15. of the second stage broth were added to each test flask. The test flasks were incubated at 30C. for three days on a rotary shaker at 220 rpm; the rotary shaker having a 1 inch throw. After the incubation period all the flasks were adjusted to the initial volume with distilled water. 10 ml. samples were centrifuged for separation of the residual hydrocarbon cells and the aqueous fractions. The cells were between the oil and aqueous phases. Dodecanedioic acid assay of the aqueous layer was done by a thin layer chromatography. The results were that 67 millimoles of dodecanedioic acid were produced per liter of reactants in the test flask. The yield was 38 percent (mole) and conversion was 50 percent (mole).

EXAMPLE 111 Following the procedure of Example 11, 1,12- dodecanedioic acid was prepared from dodecane in the presence of Solotrol 170 (same as in Example 11) using the bacteria shown in Table I. The results of the runs are shown in Table l.

mM millimoles EXAMPLE IV Following the procedure of Example ll, 1,12- dodecanedioic acid was prepared from dodecane in the presence of a blend of n-alkanes of 15 to 24 carbons rather than the Solotrol 170 using the bacterium shown in Table II. The results of the runs are shown in Table ll.

TABLE II l,l2-DODECANEDIOIC ACID Conver- STRAIN mM/ Yield sion liter (mole) (mole) Corynebacterium sp. G 11-25, ATCC 2l744 53 25 58 Corynebacterium sp. 234-30, ATCC 21745 102 34 85 Coryncbacterium sp. 11-32. ATCC 21746 82 26 96 97 31 95 76 27 83 62 29 58 mM millimoles We claim:

1. In a process for the production of alpha,omegaalkanedioic acid of to 14 carbon atoms in a mixture comprising n-alkane of 10 to 14 carbon atoms, culture medium and a mutant of Corynebacterium capable of producing alpha,omega-alkanedioic acid at high yield and conversion, the improvement which comprises operating the process in the presence of an inducer selected from the class consisting of a blend comprising substantially branched saturated aliphatic hydrocarbons of 12 to 22 carbon atoms, the blend having a boiling point of from 150 to 255C., a blend comprising n-alkanes having from to 24 carbon atoms and mixtures thereof with the provisoes that initially the ratio of the volume of the inducer to the n-alkane of 10 to 14 carbon atoms is from 3:1 to 1:3 and the volume of n-alkane of 10 to 14 carbon atoms is from 1 to 50 percent by volume of the total volume of the cultiire medium and n-alkane of 10 to 14 carbon atoms.

2. The process of claim 1 in which the inducer is a blend comprising substantially branched saturated aliphatic hydrocarbons which comprises hydrocarbons of 14 to 22 carbon atoms and which has a boiling point of from 196 to 250C.

3. The process of claim 2 in which the ratio of the volume of the inducer to the n-alkane of 10 to 14 carbon atoms is about 1:1.

4. The process of claim 3 in which the volume of nalkane of 10 to 14 carbon atoms is 5 to 20 percent of the total volume of the culture medium and n-alkane of 10 to 14 carbon atoms.

5. The process of claim 4 in which the n-alkane of 10 to 14 carbon atoms is of 12 carbon atoms.

6. The process of claim 1 in which the process to which the improvement is made comprises 1) introducing a mutated strain of Corynebacterium capable of producing at high yield and conversion alpha,omegaalkanedioic acid of 10 to 14 carbon atoms into a composition which has a pH of from 6 to 9 and which comprises 1 to 50 percent by volume of n-alkane of 10 to 14 carbon atoms and a complemental amount of culture medium; 2) maintaining the composition at a temperature of between about 20 to 45C. for 48 to 96 hours; and 3) separating the alpha,omega-alkanedioic acid from the resulting mixture.

7. The process of claim 5 in which the culture medium is an aqueous culture medium comprising (1) 3 to 35 grams per liter of a member selected from the class consisting of alkali metal nitrate, calcium nitrate, magnesium nitrate, strontium nitrate, beryllium nitrate and mixtures thereof; (2) 0.3 to 10 grams per liter of alkali metal phosphate: (3) l to 15 grams per liter of a member selected from the class consisting of alkali metal acetate, calcium acetate, magnesium acetate, strontium acetate, beryllium acetate and mixtures thereof; and (4) a nutrient source. 

2. The process of claim 1 in which the inducer is a blend comprising substantially branched saturated aliphatic hydrocarbons which comprises hydrocarbons of 14 to 22 carbon atoms and which has a boiling point of from 196* to 250*C.
 3. The process of claim 2 in which the ratio of the volume of the inducer to the n-alkane of 10 to 14 carbon atoms is about 1:
 4. The process of claim 3 in which the volume of n-alkane of 10 to 14 carbon atoms is 5 to 20 percent of the total volume of the culture medium and n-alkane of 10 to 14 carbon atoms.
 5. The process of claim 4 in which the n-alkane of 10 to 14 carbon atoms is of 12 carbon atoms.
 6. The process of claim 1 in which the process to which the improvement is made comprises 1) introducing a mutated strain of Corynebacterium capable of producing at high yield and conversion alpha,omega-alkanedioic acid of 10 to 14 carbon atoms into a composition which has a pH of from 6 to 9 and which comprises 1 to 50 percent by volume of n-alkane of 10 to 14 carbon atoms and a complemental amount of culture medium; 2) maintaining the composition at a temperature of between about 20* to 45*C. for 48 to 96 hours; and 3) separating the alpha,omega-alkanedioic acid from the resulting mixture.
 7. The process of claim 5 in which the culture medium is an aqueous culture medium comprising (1) 3 to 35 grams per liter of a member selected from the class consisting of alkali metal nitrate, calcium nitrate, magnesium nitrate, strontium nitrate, beryllium nitrate and mixtures thereof; (2) 0.3 to 10 grams per liter of alkali metal phosphate: (3) 1 to 15 grams per liter of a member selected from the class consisting of alkali metal acetate, calcium acetate, magnesium acetate, strontium acetate, beryllium acetate and mixtures thereof; and (4) a nutrient source. 